Fundamental gaps in our understanding of HIV reservoirs preclude a precisely targeted approach to eradication. HIV is neither eliminated nor often controlled by the human immune system, and the immunologic defect(s) responsible for this lack of control are unknown. Two key issues in understanding HIV persistence during therapy are (1) characterizing the relative contributions of active replication and chronic reservoirs to HIV persistence and (2) describing the cell type and activation state of cells chronically infected with HIV, which may identify new approaches to identify, disrupt, and eliminate persistently infected reservoirs. Ultimately, these approaches must be evaluated in vivo in clinical trials. ___Previously, we developed a standard clinical approach to investigating persistence using a strategy of frequent sampling to quantify HIV viremia during an extended baseline evaluation, followed by intensive monitoring of viremia during a targeted intervention, and evaluation of a post-treatment period. This strategy is optimized to quantify the level and variability of viremia prior to intervention, detect the presence of statistically significant changes during intervention, and determine whether any persistent effects or rebound occur after intervention is discontinued. ___In this project, we have developed a number of new studies to explore interventions using the intensification design. These studies will assess the initial efficacy of potential approaches and provide a wealth of patient-derived material to investigate the source and characterize the mechanisms of HIV persistence. ___We have also developed a series of clinical studies to investigate the relative effects of generalized and specific immune activation on persistent HIV viremia and latency reactivation. We hypothesize that HIV reactivation from latently infected cells can occur as a consequence of nonspecific immune activation or activation from anamnestic responses from specific antigenic stimulation. ___To investigate the role of generalized immune activation in HIV persistence, we are investigating processes that lead to nonspecific immune activation in HIV-infected individuals. One prominent mechanism described for such activation is the translocation of bacterial cell products across the gastrointestinal barrier into the systemic circulation; the presence of these bacterial products leads to generalized immune activation and may contribute to activation of immune cells, including HIV-infected cells, leading to persistent viremia. We are investigating the effects of the non-absorbable antibiotic rifaximin, which reduces bacterial flora within the gastrointestinal tract. In a multisite randomized, double-blind, placebo-controlled crossover study, we are determining whether reductions in bacterial flora in the gut reduce translocation of bacterial cell products, cellular immune activation, and the level of persistent viremia in patients with viral RNA levels suppressed below 50 copies/ml plasma on combination antiretroviral therapy (ART). The protocol A Double Blind Randomized Placebo Controlled Study Examining the Effects of a Non-absorbable (Rifaximin) Antibiotic on the Chronic Immune Activation Observed in HIV-infected Subjects (13-I-0062) is open at NIH and at two additional sites, Walter Reed National Military Medical Center (A. Ganesan) and University of Pittsburgh (D. McMahon). The NIH is the coordinating center for this study. These studies will directly address the role of chronic immune activation in persistent viremia. ___In the past year, we successfully applied for new Bench-to-Bedside funds to investigate the sources of HIV persistence. In the new study Localizing Reservoirs of HIV Persistence in Lymphoid Tissue, we will be collaborating with B. Wood, A. Venkatesan, and D. Hammoud from Interventional Radiology to localize and biopsy metabolically active tissue obtained using PET scanning and a specific targeting biopsy technique pioneered by Drs. Wood and Venkatesan. ___A second possible source of immune activation to drive HIV production is potent antigenic stimulation resulting in activation of memory cells containing latent HIV, resulting in increases in virus production. At any given time, a subset of latently infected cells may be responding to cognate antigens and undergoing activation, resulting in virus expression contributing to persistent HIV viremia. We are investigating whether administration of a common recall antigen results in increases in persistent viremia. Our hypothesis is that interventions that expose the immune system to recall antigens will result in increases in plasma HIV RNA levels. We investigated the feasibility of studying specific antigenic stimulation and HIV viremia by determining the effects of the administration of seasonal and pandemic influenza vaccine on levels of persistent viremia. The 2009-2010 influenza season was characterized by circulation of H3N2, H1N1 (seasonal), B, and H1N1 (swine/pandemic) influenza viruses. We performed a pilot substudy of the natural history study of HIV infection 95-I-0072, and obtained additional phlebotomy from patients with suppressed viremia undergoing ART who were receiving influenza vaccination. ___We are also determining the relative effects of cellular expansion on the levels of persistent HIV viremia and latency reactivation. The presence of persistent plasma clones during persistent viremia during therapy suggests that viremia may in part derive from cells undergoing cell division, expanding the reservoir of infected cells without affecting viral genetic diversity. These and other data suggest that cells with proliferative capacity are potential sources of HIV reservoirs. We are collaborating with R. Yarchoan (NCI HIV and AIDS Malignancy Branch) in a study of administration of cytotoxic chemotherapy and local radiation therapy for HIV-associated anal neoplasms (11-C-0129). Adapting the intensification model, patients with suppressed viremia are undergoing additional phlebotomy to obtain plasma and PBMC before, during, and after cycles of chemotherapy with the alkylating agent 5-fluorocuracil and DNA crosslinking agent mitomycin C (N=15). In addition, patients will be sampled during and following subsequent periods of radiation. Our hypothesis is that, by suppressing cell division, alkylating and crosslinking agents will result in decreases in HIV viremia, especially in patients with PPC, but local radiation will not result in changes in HIV RNA. In addition, we will analyze HIV population genetics from plasma, PBMC, and from GALT obtained from regions proximal and distant to the tumor tissue to determine whether changes in viremia can be mapped to specific reservoirs. Protocol 11-C-0129 is open at NIH and is currently accruing patients. ___Innate immune responses have critical effects on the course of HIV infection, and we are also determining the effects of innate immune modulator interferon alpha 2b on levels of persistent HIV viremia and latency reactivation. Our hypothesis is that HIV plasma viremia will be reduced early during interferon therapy, with consequent increases in cell-associated HIV RNA but not HIV DNA, due to tetherin-induced effects preventing HIV release from cells. With time on interferon, we anticipate a decline in infected cell number as infected cells with increased surface HIV will be more easily identified by immune cells. This trial (11-I-0057, Effect of Interferon Alpha 2b Intensification on HIV-1 Residual Viremia in Individuals Suppressed on Antiretroviral Therapy) is a collaborative study with D. McMahon (University of Pittsburgh) and is IRB approved and ongoing.